A New Sterol From the Polypore Fungus Ganoderma luteomarginatum and Its Cytotoxic Activities

Abstract

A new sterol, ganolutol A (1), together with 4 known compounds 2 to 5, were isolated from the polypore fungus Ganoderma luteomarginatum collected in Myanmar. The chemical structures of the isolated compounds were determined based on extensive spectroscopic analyses in conjunction with comparisons with published data. Compounds 1 to 4 exhibited moderate cytotoxic activities against A549 (lung), MCF-7 (breast), and HeLa (cervical) human cancer cell lines, with IC₅₀ values ranging from 10.1 to 86.6 μM.

Keywords

Ganodermataceae polypore fungus ergostane cytotoxicity

Introduction

Ganoderma luteomarginatum is a rare polypore fungus belonging to the family Ganodermataceae. It is mainly distributed throughout tropical and subtropical areas including China, Laos, Myanmar, Thailand, and Vietnam.^1,2 The G luteomarginatum fruiting bodies have been utilized in traditional medicine to treat and prevent various diseases such as hypertension, diabetes, hepatitis, and cancers in China, Japan, and Korea.^3,4 The fruiting bodies are known as “Lin zhi” in Myanmar and their extracts have long been used locally for the purposes of liver protection, blood purification, and detoxification, as well as tumor treatment.⁵ A few previous phytochemical investigations of ethanol extracts of G luteomarginatum fruiting bodies collected in China led to the isolation of 12 lanostane-type triterpene acids, a pair of alkaloid enantiomers, and 9 lanostane triterpenoids.^1,6,7 Among them, lanostane triterpenoids such as (17Z)-3β,7β,15β-trihydroxy-11,23-dioxolanost-8,17(20)-dien-26-oate, (20E)-15β-hydroxy-3,7,11,23-tetraoxolanost-20(22)-en-26-oate, and (5α,24E)-3β-acetoxyl-26-hydroxylanosta-8,24-dien-7-one exhibited strong cytotoxicities against gastric HGC-27, cervical HeLa, and lung A549 human cancer cell lines.^1,6 In the course of our search for cytotoxic compounds from traditional folk medicines in Myanmar, phytochemical investigations of the ethyl acetate-soluble fraction of a 70% ethanolic extract of the Myanmar G luteomarginatum fruiting bodies revealed the presence of one new ergostane-type steroid and four known compounds. Herein, we report the isolation and structure elucidation of the compounds and their cytotoxicities.

Results and Discussion

Various chromatographic procedures on the ethyl acetate-soluble fraction of the 70% ethanolic extract of G luteomarginatum led to the isolation of 5 compounds, including 1 new ergostane-type steroid, named ganolutol A (1) and 4 known compounds (Figure 1). The known compounds were identified as ergosterol (2),⁸ ergosterol peroxide (3),⁹ (3β,5α,6β,22E)-6-methoxyergosta-7,22-diene-3,5-diol (4),¹⁰ and 4-hydroxy-2′,4′-dimethoxydihydrochalone (5),¹¹ by comparison of their 1D NMR spectroscopic data with those in the literature.

Figure 1.

Structures of isolated compounds 1 to 5 from Ganoderma luteomarginatum.

Compound 1 was obtained as a white amorphous powder. Its molecular formula was determined as C₂₈H₄₄O₅ from HR-ESI-MS data (m/z 459.3108, [M-H]⁻, calcd. for C₂₈H₄₃O₅, 459.3116) and interpretation of the ¹³C NMR data, which indicated 7 degrees of unsaturation (Supplementary Figures S7). The IR spectrum showed absorption bands corresponding to hydroxy (3418 cm⁻¹) and olefinic (1633 cm⁻¹) functionalities. The ¹H NMR data (Table 1 and Supplementary Figure S1) showed characteristic signals corresponding to 2 tertiary methyl protons [δ_H 0.63 (s, H₃-18), 1.37 (s, H₃-19)], 4 secondary methyl protons [δ_H 1.03 (d, J = 6.4 Hz, H₃-21), 0.87 (d, J = 6.8 Hz, H₃-26), 0.88 (d, J = 6.8 Hz, H₃-27), 0.96 (d, J = 6.8 Hz, H₃-28)], 3 olefinic protons [δ_H 5.95 (t, J = 3.6 Hz, H-7), 5.23 (dd, J = 15.4, 7.2 Hz, H-22), 5.22 (dd, J = 15.4, 8.0 Hz, H-23)], and 2 oxygenated methine protons [δ_H 4.47 (m, H-3), 4.64 (t, J = 3.6 Hz, H-6)]. The ¹³C NMR data (Table 1 and Supplementary Figures S2 and S4) revealed 28 carbon signals, consisting of 2 oxygenated methine carbons [δ_C 66.6 (C-3), 86.4 (C-6)], 2 oxygenated carbons [δ_C 87.7 (C-5), 85.4 (C-9)], 2 quaternary carbons [52.2 (C-10), 42.6 (C-13)], 4 olefinic carbons [δ_C 121.3 (C-7), 143.0 (C-8), 136.3 (C-22), 132.8 (C-23)], 5 methine carbons [δ_C 53.1 (C-14), 56.0 (C-17), 41.1 (C-20), 43.5 (C-24), 33.8 (C-25)], 7 methylene carbons [δ_C 29.6 (C-1), 33.2 (C-2), 37.3 (C-4), 24.0 (C-11), 37.4 (C-12), 23.7 (C-15), 28.9 (C-16)], and 6 methyl carbons [δ_C 12.1 (C-18), 17.6 (C-19), 21.7 (C-21), 20.3 (C-26), 20.6 (C-27), 18.3 (C-28)].

Table 1.

¹H (400 MHz) and ¹³C (100 MHz) NMR Data of 1 in pyridine-d₅.

	1			Reference 1′
Position	δ_H (J in Hz)	δ _C	Type	δ _C
1α	1.43, m	29.6	CH₂	28.7
1β	2.21, m
2α	2.22, m	33.2	CH₂	31.7
2β	1.77, m
3	4.47, m	66.6	CH	66.8
4α	2.86, dd (14.8, 11.2)	37.3	CH₂	34.8
4β	2.76, dd (14.8, 3.6)
5		87.7	C	86.6
6	4.64, t (3.6)	86.4	CH	72.2
7	5.95, t (3.6)	121.3	CH	122.5
8		143.0	C	141.8
9		85.4	C	84.7
10		52.2	C	51.0
11α	1.80, m	24.0	CH₂	28.0
11β	1.84, m
12α	1.34, m	37.4	CH₂	34.7
12β	1.89, m
13		42.6	C	41.9
14	2.29, m	53.1	CH	52.1
15α	1.71, m	23.7	CH₂	23.2
15β	1.36, m
16α	1.71, m	28.9	CH₂	28.1
16β	1.93, m
17	1.19, m	56.0	CH	55.5
18	0.63, s	12.1	CH₃	11.7
19	1.37, s	17.6	CH₃	17.3
20	2.00, m	41.1	CH	39.5
21	1.03, d (6.4)	21.7	CH₃	21.1
22	5.23, dd (15.4, 7.2)	136.3	CH	135.2
23	5.22, dd (15.4, 8.0)	132.8	CH	132.2
24	1.88, m	43.5	CH	42.8
25	1.48, m	33.8	CH	33.1
26	0.87, d (6.8)	20.3	CH₃	19.7
27	0.88, d (6.8)	20.6	CH₃	20.0
28	0.96, d (6.8)	18.3	CH₃	17.6

The above NMR data closely resembled those of the ergostane-type steroid isolated from Panellus serotinus, 5α,9α-epidioxy-(22E)-ergosta-7,22-diene-3β,6β-diol (1′), with a lower molecular weight than that of 1 by 16-mass units.¹² The main difference between 1 and 1′ was the oxygenated methine resonance at C-6, where the oxygenated methine proton and carbon signals were shifted upfield from δ_H 4.26 (m) and δ_C 72.2 in 1′ to δ_H 4.64 (t, J = 3.6 Hz) and δ_C 86.4 in 1, respectively. Considerations of the differences between the NMR data of 1 and 1′ and the 16-mass unit higher molecular weight of 1 than 1′ suggested that compound 1 is a hydroperoxy analog of 1′ at C-6. The ¹H-¹H COSY correlations of H-14 (δ_H 2.29)/H-15β (δ_H 1.36)/H-16β (δ_H 1.93)/H-17 (δ_H 1.19)/H-20 (δ_H 2.00)/H₃-21 (δ_H 1.03)/H-22 (δ_H 5.23) and the HMBC correlations from H-11β (δ_H 1.84) to C-8 (δ_C 143.0) and C-13 (δ_C 42.6), from H-12β (δ_H 1.89) to C-9 (δ_C 143.0) and C-14 (δ_C 53.1), from H₃-28 to C-23, C-24, and C-25, and from H₃-26 and H₃-27 to C-24 and C-25 (Figure 2 and Supplementary Figures S3 and S5) indicated that 1 possessed the same planar structures as those of 1′ at the C and D rings and the side-chain unit at C-17. The ¹H-¹H COSY cross-peaks of H₂-1 (δ_H 1.43, 2.21)/H₂-2 (δ_H 2.22, 1.77)/H-3 (δ_H 4.47)/H₂-4 (δ_H 2.86, 2.76) and the HMBC correlations from H-4α (δ_H 2.86) to C-5 (δ_C 87.7) and C-10 (δ_C 52.2) and from H₃-19 to C-1 (δ_C 29.6), C-5 (δ_C 87.7), and C-9 (δ_C 85.4) supported the presence of the hydroxy and methyl groups at C-3 and C-19, respectively, as well as the peroxide bridge between C-5 and C-9, as in the case of 1′ (Figure 2). The HMBC correlations from H₃-18 (δ_H 0.63) to C-12 (δ_C 37.4), C-14 (δ_C 53.1), and C-17 (δ_C 56.0) confirmed the presence of the methyl group at the same C-18 position as that of 1′. Furthermore, the ¹H-¹H COSY cross-peak of H-6 (δ_H 4.64)/H-7 (δ_H 5.95) and the HMBC correlations from H-7 (δ_H 5.95) to C-9 (δ_C 85.4) and C-14 (δ_C 53.1) indicated the existence of the oxygenated group at C-6, suggesting that a hydroperoxy group was attached to C-6.

Figure 2.

Key HMBC (arrows) and ¹H-¹H COSY (bold lines) correlations of 1.

The relative configuration of 1 was assigned from the NOESY spectrum analyses and the coupling constant value (Figure 3 and Supplementary Figure S6). The NOESY correlations starting from H₃-18 (δ_H 0.63) to H-1β (δ_H 2.21) via H-11β (δ_H 1.84)/H₃-19 (δ_H 1.37) and from H₃-18 (δ_H 0.63) to H-4β (δ_H 2.76) via H-11β/H₃-19/ H-2β (δ_H 1.77) indicated that the methyl groups at C-10 and C-13, H-1β, and H-4β were β-oriented. The NOESY correlations from H-1α (δ_H 1.43) to H-3 (δ_H 4.47) and from H-4α (δ_H 2.86) to H-6 (δ_H 4.64) and the lack of the NOESY correlation between H-6 and H₃-19 confirm the α-orientations of H-3 and H-6 as well as β-orientations of the hydroxy group at C-3 and the hydroperoxy group at C-6. Furthermore, the NOESY correlations from H-12β (δ_H 1.88) to H-14 (δ_H 2.29) and H-14 to H-17 (δ_H 1.19) indicated the α-orientations of H-14 and H-17. The Δ22-double bond was elucidated to have the E configuration, based on the ¹H-¹H coupling constant (J = 15.4 Hz) between H-22 and H-23.

Figure 3.

Key NOESY correlations (dashed arrows) of 1.

Primary CD calculation analyses of the (3S,5R,6R,9R,10R,13R,14R,17R)-1 model with each set of 20S and 24S, 20S and 24R, 20R and 24S, and 20R and 24R indicated that the side-chain did not affect the CD spectra, suggesting that the absolute configurations at C-20 and C-24 could not be determined by CD analysis (Supplementary Figure S8). Thus, the absolute configuration of 1 was elucidated by comparisons of its CD spectrum with the calculated CD spectra of (3S,5R,6R,9R,10R,13R,14R,17R,20R,24R)-1 and its enantiomer (Figure 4) and the chemical shift values between 1 and ergosterol (2), as well as from a biogenetic viewpoint. Compound 1 showed positive and negative Cotton effect values at 265 and 218 nm, respectively, in the CD spectrum. The CD spectrum was closely similar to that of the (3S,5R,6R,9R,10R,13R,14R,17R,20R,24R)-1 model, revealing that 1 possessed the same absolute configurations as those of 2 at C-3, C-9, C-10, C-13, C-14, and C-17. In light of the same absolute configurations at these positions between 1 and 2 and the quite similar carbon chemical shift values between these compounds, the remaining absolute configurations of C-20 and C-24 in 1 were biogenetically determined to be the same R and R as those in 2, respectively. Hence, compound 1 was assigned to be (3S,5R,6R,9R,10R,13R,14R,17R,20R,24R)-5,9-epidioxy-3-hydroxy-(22E)-ergosta-7,22-dien-6-hydroperoxide, and was named ganolutol A.

Figure 4.

Calculated and experimental ECD spectra of 1.

The ethyl acetate-soluble fraction and the isolated compounds 1 to 5 were assessed for their cytotoxicities against A549, MCF-7, and HeLa human cancer cell lines. Compounds 1 to 4 did not show any cytotoxicities against the tested cancer cell lines as shown in Table 2 and Supplementary Figure S9.

Table 2.

Cytotoxic Activities of Compounds 1 to 5.

	IC₅₀ (μg/mL, μM)
Sample	A549	MCF-7	HeLa
EtOAc^c	47.6^a	65.6^a	55.2^a
1	63.1^b	51.6^b	86.6^b
2	19.1^b	69.3^b	20.2^b
3	19.3^b	18.1^b	10.1^b
4	18.7^b	19.6^b	26.9^b
5	64.8^b	124.3^b	95.7^b
5-fluorouracil^d	5.0^b	3.2^b	18.3^b

Note: ^aμg/mL.

μM.

Ethyl acetate-soluble fraction.

Positive control.

Conclusions

A new sterol, ganolutol A (1), and four known compounds 2 to 5 were isolated from the polypore fungus, G luteomarginatum. Compounds 1 to 4 did not show any cytotoxicity against the tested A549, MCF-7, and HeLa cancer cell lines.

Experimental Section

General Experimental Procedures

NMR spectra were recorded on a JEOL ECA400II spectrometer. Pyridine-d₅ and CDCl₃ were used to solve all isolated compounds. δ_H 8.74 and δ_C 150.35 for pyridine-d₅ and δ_H 7.26 and δ_C 77.0 for CDCl₃ of the residual solvent peaks were used to calibrate the signals. HR-ESI-MS data were obtained with a Shimadzu LCMS-IT-TOF spectrometer. Optical rotation and CD measurements were performed on a JASCO P2100 polarimeter and a JASCO J-805 spectropolarimeter, respectively. UV and IR spectra were measured on an Implen NP80 nanospectrometer and a JASCO FT/IR-460 Plus spectrometer (KBr pellets), respectively. Silica gel 60N (spherical, neutral, 40-50 μm, Kanto Chemical) and Cosmosil 75C18-OPN (Nacalai Tesque) were used to perform normal phase and reverse phase open column chromatography, respectively. Thin-layer chromatography (TLC) was performed on silica gel GF₂₅₄ pre-coated plates (Merck). The compounds were detected under a UV lamp (254 and 365 nm) and by spraying with a p-anisaldehyde stain solution followed by heating at 170 °C for 10 min in a drying cabinet.

Fungal Material

The fruiting bodies of G luteomarginatum were purchased from the local medicinal market in Aungban, Shan State of Myanmar and were identified by Dr Ni Lar Cho, a botanist at the Department of Botany, Meiktila University. A voucher specimen (31185) has been deposited in the Museum for Materia Medica, Analytical Research Center for Ethnomedicines, Institute of Natural Medicine, University of Toyama, Japan.

Extraction and Isolation

The fruiting bodies of G luteomarginatum (1.49 kg) were chipped and extracted with 70% ethanol under sonication (12 L, 3 h, × 5) at 30 °C after drying. The solvent was then evaporated, and the resultant 70% ethanolic extract (45.9 g) was suspended in water and partitioned with ethyl acetate to give an ethyl acetate-soluble fraction (31.5 g). The ethyl acetate-soluble fraction was chromatographed on a silica gel column eluted with n-hexane − ethyl acetate (90:10 to 0:100) and ethyl acetate − methanol (90:10 to 0:100) to give eleven fractions (F₁₋₁₁). Fraction F₄ (2.8 g) was rechromatographed on a silica gel column eluted with n-hexane − dichloromethane − ethyl acetate (40:40:20 to 0:50:50) to afford 4 subfractions, F₄₋₁ (180 mg), F₄₋₂ (166 mg), F₄₋₃ (54 mg), and F₄₋₄ (2.4 g). Subfraction F₄₋₂ (166 mg) was subjected to a silica gel column eluted with n-hexane − ethyl acetate (80:20 to 0:100) to afford 2 (12.0 mg) and 3 (16.0 mg). Purification of subfraction F₄₋₃ (54 mg) on a silica gel column eluted with n-hexane − ethyl acetate (60:40 to 50:50) gave 4 (11 mg). Subfraction F₄₋₄ (2.4 g) was rechromatographed on a silica gel column eluted with n-hexane − ethyl acetate (60:40 to 50:50) to give 7 subfractions (F₄₋₄₋₁ to F₄₋₄₋₇). Subfraction F₄₋₄₋₂ was subjected to chromatography on a silica gel column eluted with n-hexane − ethyl acetate (66:34 to 50:50) to afford 1 (10.5 mg). Fraction 5 (1.6 g) was subjected to chromatography on an ODS column, eluted with water − methanol (100:0 to 0:100) to give 7 subfractions (F₅₋₁ to F₅₋₇). Compound 5 (7.7 mg) was obtained from the subfraction F₅₋₃ by chromatography on a silica gel column, eluted with n-hexane − ethyl acetate (66:34 to 50:50).

Ganolutol A (1): white amorphous powder; [α]₂₅^D = + 31.5 (c 0.1, MeOH); UV (MeOH) λ_max (log ε) 209 (3.62), 218 (3.64) nm; CD (MeOH) λ_max (Δε) 218 (−6.30), 265 ( + 1.49) nm; IR (KBr) ν_max 3418, 1720, 1633, 1457, 1388, 1103, 840 cm⁻¹; ¹H and ¹³C NMR data, see Table 1; negative HR-ESI-MS m/z [M-H]⁻ 459.3108 (calcd. for C₂₈H₄₃O₅, 459.3116).

ECD Calculations

The conformational search was conducted as previously described with some modifications.¹³ The Avogadro 1.2 program was used to obtain the MMFF94 molecular force field. The optimization of all possible conformers at the B3LYP level of theory was performed by using the 6-31G(d) basis set, and the minimum energy of the structure was ensured by calculating their optimized geometries. The geometries used for the ECD calculations were obtained from Gaussian 16 at the B3LYP/6-31G(d) level. GaussSum using a half bandwidth of 0.2 eV was used to generate the ECD curves. All spectra of the lowest energy conformations were averages.

Cell Culture and Cytotoxicity Assay

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay¹⁴ was used to evaluate the cytotoxicities of the isolated compounds in cell lines. The human cancer cell lines (A549, MCF-7, and HeLa) were cultured in α-Minimum essential medium with phenol red and L-glutamine (α-MEM, Wako).^15–20 A 1% antibiotic antimycotic solution (Sigma) and 10% fetal bovine serum (FBS; Sigma) were supplemented to all media. For the MCF-7 cells, 1% 1 mM sodium pyruvate (Gibco) and 1% 0.1 mM nonessential amino acids (NEAA, Gibco) were supplemented in the growth medium. Briefly, each cancer cell line was seeded at 2 × 10³ cells per well in 96-well plates and incubated for 24 h in the respective medium at 37 °C, under a 5% CO₂ and 95% air atmosphere. The cells were then washed with phosphate-buffered saline (PBS), and the tested samples were added to each well at 6 concentrations (6.25, 12.5, 25.0, 50.0, 100, and 200 μM). After a 72 h incubation, the cells were washed with PBS, and to each well, 100 μL of medium containing 10 µL of the MTT solution (5 mg/mL) was added and incubated for 3 h. Subsequently, each well was monitored to calculate cell viability at 570 nm, using the following equation (each set of cell viability at each concentration was the mean value of the data from 3 wells):

(%) Cell viability = 100 \times {[Ab s_{(test sample)} - Ab s_{(blank)}] / [Ab s_{(control)} - Ab s_{(blank)}]}

GraphPad Prism 8.0 software was used to evaluate the IC₅₀ value of the cytotoxicity on a sigmoid dose–response model. 5-Fluorouracil (Wako) was used as a positive control.

Supplemental Material

sj-docx-1-npx-10.1177_1934578X221098852 - Supplemental material for A New Sterol From the Polypore Fungus Ganoderma luteomarginatum and Its Cytotoxic Activities

Supplemental material, sj-docx-1-npx-10.1177_1934578X221098852 for A New Sterol From the Polypore Fungus Ganoderma luteomarginatum and Its Cytotoxic Activities by Zin Paing Htoo, Takeshi Kodama, Nwet Nwet Win, Naotaka Ikumi, Ken-ichi Shiokawa and Hiroyuki Morita in Natural Product Communications

Footnotes

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

Ethical Approval

Not applicable, because this article does not contain any studies with human or animal subjects.

Informed Consent

Not applicable, because this article does not contain any studies with human or animal subjects.

ORCID iD

Hiroyuki Morita

Trial Registration

Not applicable, because this article does not contain any clinical trials.

Supplemental Material

Supplemental material for this article is available online.

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